Throughout this application various publications are referred to in square brackets. Full citations for these references may be found at the end of the specification. The disclosures of these publications, and of all patents, patent application publications and books referred to herein, are hereby incorporated by reference in their entirety into the subject application to more fully describe the art to which the subject invention pertains.
Archived human specimens, with known clinical follow-up, represent a valuable resource, particularly for retrospective molecular studies and identification of biological markers that might be useful for risk prediction of disease or prognosis [1].
Recent studies have demonstrated that nucleic acids recovered from archived specimens are suitable for a variety of downstream genetic (genomic and transcriptomic) and epigenetic analyses [1]. Genomic DNA recovered from archived specimens, while degraded, can be analyzed by polymerase chain reaction (PCR) [2,3], array comparative genomic hybridization (CGH) [4], massively parallel sequencing [5], and methylation assays [6-8].
Contrastingly, messenger RNA molecules recovered from formalin-fixed paraffin-embedded (FFPE) specimens display a large extent of degradation, and thus many studies have aimed at demonstrating their suitability for molecular analyses and specific protocols have been established for quantitative reverse transcription PCR (qRT-PCR) [2,9], high-throughput gene expression [1,10-12], and even massive parallel sequencing [13,14]. Interestingly, microRNAs, due to their small size, remain intact throughout the processes of formalin-fixation and RNA extraction, and they can be reliably studied in FFPE specimens [15,16].
Protocols for genomic DNA or total RNA extractions, from FFPE specimens, have been well documented and made available as reliable commercial kits [17,18]. In general, tissue sections are deparaffinized in a non-polar solvent: xylene, Hemo-D (dlimonene), or citrisolv and then subjected to proteinase-K digestion, usually short (15 minutes to overnight) for RNA, to minimize degradation, but extended (for up to 48 h) for DNA isolation [1]. To increase DNA purity, exposure to high-temperature (95-98° C.), in an alkaline buffer, has been shown to allow removal of DNA/protein cross-links, a denaturing step however that cannot be used during RNA isolation [19-22]. To avoid cross-contamination between these two types of nucleic acids, an RNase or DNAse treatment for DNA or RNA purification, respectively, is added prior to either a solvent separation (TRIzol, phenol/chloroform) or a silica-based column purification [11,18]. To increase RNA quality, a final step consists of heat-treatment at 70° C. for up to 60 minutes, in a Tris-EDTA (1× TE) or citrate-based buffer, to remove chemical modifications (methylol groups) acquired during formalin-fixation [23,24]. Based on these different biochemical requirements, DNA and RNA have routinely been extracted separately.
The recovery of genomic DNA and total RNA from the same specimen would have the advantage of providing matched nucleic acid fractions, from the same cells, which would be extremely valuable for validations as well as for integrative studies. Maximizing DNA and RNA retrieval from a single specimen might also be very useful when using tissues that are of limited availability.
The present invention addresses the need for simultaneously recovering RNA and DNA from the same specimen sample, including FFPE archival samples, fresh samples, sera and serum exosomes.